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Systematic investigation of the toxic mechanism of difenoconazole on protein by spectroscopic and molecular modeling
Pesticide Biochemistry and Physiology 105 (2013) 155–160
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Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Systematic investigation of the toxic mechanism of difenoconazole on protein by spectroscopic and molecular modeling Ying Li, Xueru Ma, Guanghua Lu ⇑ Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes of Ministry of Education, College of Environment, HoHai University, NanJing, JiangSu Province 210098, China
a r t i c l e
i n f o
Article history: Received 29 May 2012 Accepted 31 December 2012 Available online 20 January 2013 Keywords: Difenoconazole Protein Molecular modeling Multi-spectroscopy
a b s t r a c t In order to better understand the toxic effects of difenoconazole, we performed multi-spectroscopic measurements to quantify the interaction of difenoconazole with human serum albumin. The toxicity mechanism was predicted through molecular modeling, and the binding parameters were confirmed using a series of spectroscopic methods, including UV–vis absorption spectroscopy, Fourier transform infrared spectroscopy, circular dichroism spectroscopy and fluorescence spectroscopy. Alteration of secondary structure of protein was evaluated, then the specific binding site in protein was identified and binding constants were determined. The molecular modeling study and thermodynamic analysis suggested that difenoconazole could bind HSA through the hydrophobic force, electrostatic interaction and hydrogen bond. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Difenoconazole (structure shown in Fig. 1) was developed by Switzerland Syngenta Corporation, IUPAC name: cis,trans-3chloro-4-[4-methyl-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan2-yl] phenyl-4-chlorophenylether)phenyl-4-chlorophenyl ether, belonging to the triazole group of pesticides. As a kind of systemic sterol demethylation inhibitor (DMI), difenoconazole has a good ability to interfere with the mycelial growth and inhibit the spore germination of pathogens that ultimately results in inhibiting fungal growth [1]. Difenoconazole is extensively used in a wide range of crops in many countries for its good control of various fungal diseases. However most of pesticides are not completely degraded after application, their metabolites and some unchanged forms of these compounds are excreted and subsequently enter the ecosystem. Although the triazole fungicides have shorter half-lives and lower bioaccumulation than the organochlorine pesticides, possible detrimental effects on the ecosystem and human health were also existed. For this reason, knowledge of the interaction mechanisms between difenoconazole and plasma protein is of crucial importance for us to understand its possible danger to human body. On the basis of the motivations described above, we settled on an initial focus on difenoconazole binding to basic human plasma protein, and understanding the toxic response. Human serum albumin (HSA) is the most abundant protein in the blood, which is ⇑ Corresponding author. E-mail address: [email protected] (G. Lu). 0048-3575/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2012.12.010
globular protein composed of a single polypeptide chain of 585 amino acid residues. It is characterized by high a-helical content and a large number of disulfide bonds, and serves in transportation and disposition of many endogenous substances and exogenous compounds, including drugs and xenobiotics [2–7]. A wide range of photophysical techniques, such as fluorescence quenching, circular dichriosm (CD) and Fourier transform infrared (FT-IR) are used in our study to characterize various aspects of the protein–difenoconazole interaction and these techniques are briefly described in Section 2. These measurement methods allow for determination of a number of basic properties: the binding constant of proteins with difenoconazole, changes in protein conformation. Specifically, fluorescence quenching measurements give information about the protein–difenoconazole binding kinetics and equilibrium and protein conformational change, CD and FTIR informs about changes in protein structure upon binding, and molecular modeling investigate the mechanism of the reaction. The use of these combined methods should provide a general perspective of how difenoconazole affects the nature of protein binding and the extent to which these effects are protein specific. These supporting information are very important for food safety and human health when using triazole antifungal agent. 2. Materials and methods 2.1. Materials Difenoconazole was of chromatographic grade, and purchased from Dr. Ehrenstorfer Company (Germany). Human Serum
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O
Cl
Cl
O
experiments were performed at four different temperatures (296, 303, 310 and 317 K). O
N N N
CH 3 Fig. 1. Chemical structure of difenoconazole.
Albumin (HSA, 70024-90-7, 98% purity) was purchased from Sigma Chemical Company. It was used without further purification and its molecular weight was assumed to be 66,478 to calculate molar concentrations. NaCl (1.0 mol/L) solution was used to maintain the ionic strength at 0.1. Buffer consists of Tris (0.2 mol/L) and HCl (0.1 mol/L), and the pH was adjusted to 7.40. A HSA solution of 1.5 105 mol/L was prepared using the Tris–HCl buffer solution and kept in dark at 4 °C. The stock solution of difenoconazole (1.0 103 mol/L) was prepared in absolute ethanol. All the other reagents were of analytical grade and doubly distilled water was used throughout the experiment.
2.2. Apparatus and methods 2.2.1. Multi-spectroscopic measurements Infrared spectroscopy were recorded at 296 K using a Nicolet Nexus 670 FT-IR Spectrometer (USA) equipped with a Geranium attenuated total reflection (ATR) accessory, a deuterated triglycine sulphate (DTGS) detector and a KBr beam splitter. All infrared spectra were taken via the attenuated total reflection method with a resolution of 4 cm1 and 128 scans. FT-IR spectrum of free HSA was acquired by subtracting the absorption of the Tris buffer solution from the spectrum of the protein solution, and a difference spectrum of HSA was obtained by subtracting the spectrum of difenoconazole from that of difenoconazole–HSA with the same concentration. The subtraction criterion was that original spectrum of the protein solution between 2200 and 1800 cm1 was featureless [8]. Secondary derivative were applied to this range respectively to estimate the number, position and width of component bands. Based on these parameters curve-fitting process was carried out by Galactic Peak solve to get the best Gaussian-shaped curves that fit the original protein spectrum. After the identification of the individual bands, the representative structure of HSA was calculated using the area of their respective component bands. Circular dichroism measurements were made on a Chriascan CD Spectrometric (Applied photophysics, UK). CD spectra were recorded in the range of 200–250 nm. The a-helical content of HSA was calculated from the molar ellipticity [h] at 208 nm using the following equation [9]:
2.2.2. Molecular modeling study The crystal structure of HSA in complex with R-warfarin was taken from the Brookhaven Protein Data Bank (entry codes 1h9z) [10]. The potential of the 3D structure of HSA was assigned based on the Amber 4.0 force field with Kollman-all-atom charges. The initial structure of difenoconazole was generated by molecular modeling software Sybyl 6.9 [11]. The geometry of the molecule was subsequently optimized to minimal energy using the Tripos force field with Gasteiger–Marsili charges, and the FlexX program was used to build the interaction modes between difenoconazole and HSA. All calculations were performed on a SGI FUEL workstation. 3. Results and discussion 3.1. Binding studies between difenoconazole and HSA using UV–vis absorption, FT-IR and CD spectrum In order to gain a better understanding in physicochemical properties of difenoconazole governing its spectral behavior and to draw relevant conclusions on the difenoconazole–HSA binding mechanism UV–vis absorption, FT-IR and CD spectroscopic measurements were performed on HSA and the difenoconazole–HSA complex. If the change of protein structure included the transforming of protein secondary structure in the complex, it can be reflected in the spectra. Fig. 2 showed the UV–vis absorption spectra of HSA in the absence and presence of difenoconazole. It can be seen from Fig. 2, the absorbance of HSA increased upon the addition of difenoconazole. At the same time, the maximum peak position of difenoconazole–HSA was shifted slightly towards lower wavelength region. This indicated the change in polarity around the tryptophan residue and the change in peptide strand of HSA molecules and hence the change in hydrophobicity [12]. These two results clearly indicated that bound to HSA and the conformation of HSA was changed. Infrared spectroscopy has long been used as a powerful method for investigating the secondary structures of proteins and their dynamics. In the IR region, the frequencies of bands due to the
a helixð%Þ ¼ fð½h208 4000Þ=ð33000 4000Þg 100 UV–vis absorption spectra were recorded on a UV-3600 UV– VIS–NIR spectrophotometer (SHIMADZU, Japan) equipped with 1.0 cm quartz cells. The wavelength range was 200–500 nm. The Fluorescence spectra were measured with a LS-50B spectrofluorophotometer (PERKIN-ELMER, USA). The fluorescence excitation wavelength was 280 nm, and the emission was read at 300– 500 nm. In a typical fluorometric titration experiment: 3.0 mL solution containing an appropriate concentration of HSA was titrated in the Tris buffer solution by successive additions of difenoconazole working solution. Titrations were done manually using micropipettes, and the fluorescence intensities were recorded at excitation wavelength of 280 nm and emission wavelength of 350 nm. All
Fig. 2. UV–vis absorption spectra of difenoconazole–HSA system. (A) [difenoconazole] = 3.32 106 M; (B) [HSA] = 1.5 106 M; (C–E) difenoconazole–HSA; [HSA] = 1.5 106 M; [difenoconazole] = 3.32 106 M, 6.62 106 M, 1.32 105 M; T = 296 K; pH = 7.4.
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amide I–III vibrations are sensitive to the secondary structure of proteins. The amide I peak position occurs in the region 1600– 1700 cm1 and the amide II band 1548 cm1 [13,14]. In the amide I region, the observed stretching frequency of C@O hydrogen bonded to NH moieties is dependent upon the secondary structure adopted by the peptide chain. Changes in the structure of the protein are reflected by changes in the component band positions of the amide I region [15]. Fig. 3 shows the FT-IR spectra of free HSA and its difenoconazole complexes in Tris–HCl buffer solution at 296 K. As shown in Fig. 3, the peak position of amide I of HSA moved from 1650 to 1640 cm1 and the peak position of amide II of HSA shifted from 1550 to 1583 cm1 after addition of difenoconazole. The changes of this peak position demonstrated that difenoconazole interacted with HSA and caused a change in the secondary structure of HSA. According to the curve-fitted results (Fig. 4), the secondary structure compositions of HSA in the absence and in the presence of difenoconazole were estimated. As shown in Fig. 4, the content of b-sheet structure composition of HSA increased from 33.15% to 39.08%, the content of b-turn structure increased from 30.72% to 33.15%, while the content of a-helix structure decreased from 36.13% to 27.77%. CD spectra of HSA exhibit characteristic features of the a-helical structure of protein with negative bands in the ultraviolet region at 208 and 222 nm [16]. Here the CD spectrum was taken in the wavelength range from 200 to 250 nm. Fig. 5 shows typical CD spectrum of HSA in the presence and absence of difenoconazole. The binding of difenoconazole complex to HSA decrease the ellipticity values in the CD spectrum, indicating the considerable changes in the protein secondary structure with the reduction of the a-helical content in HSA and the increase of the disorder structure content in the protein. The calculating results exhibited a reduction of a-helix structures from 52.98% to 48.23%. Decrease in the a-helical content in the CD spectra is consistent with the IR results. 3.2. Analysis of fluorescence quenching of HSA by difenoconazole
Fig. 4. The curve fitting amide I region with secondary structure (1700–1600 cm1). (A) HSA; (B) difenoconazole–HSA; [HSA] = 6.0 105 M; [difenoconazole] = 3.0 104 M; pH = 7.4.
An intrinsic fluorescence study was performed to evaluate changes in tertiary structure caused by reaction of HSA with difenoconazole. The effect of difenoconazole on HSA fluorescence intensity is shown in Fig. 6. The addition of difenoconazole caused a gradual decrease in the fluorescence emission intensity of HSA
Fig. 5. CD Spectra of the difennoconazole–HSA System. (A) HSA; (B) difenoconazole–HSA; [HSA] = 6.0 107 M; [difenoconazole] = 3.0 105 M; T = 296 K; pH = 7.4.
Fig. 3. FT-IR spectra and difference spectra of HSA in aqueous solution. (A) FT-IR spectrum of free HSA; (B) FT-IR difference spectrum [(HSA solution + difenoconazole solution)(difenoconazole solution)] in buffer solution; [HSA] = 6.0 105 M; [difenoconazole] = 3.0 104 M; T = 296 K; pH = 7.4.
with a conspicuous change in the emission spectra. It can be seen that a higher excess of difenoconazole led to more effective quenching of the chromophore molecule fluorescence. The strong quenching of the fluorescence clearly indicated that the binding
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The possible quenching mechanism can be deduced from the Stern–Volmer plot (Fig. 7). The classical Stern–Volmer equation [18]:
F0 ¼ 1 þ K Q s0 ½Q ¼ 1 þ K SV ½Q F
Fig. 6. Fluorescence emission spectra of difenoconazole–HSA system. (A) HSA; (B– H) difenoconazole–HSA; [HSA] = 7.5 107 M; [difenoconazole] = 3.32 106 M, 6.62 106 M, 9.90 106 M, 1.32 105 M, 1.64 105 M, 1.96 105 M, 2.28 105 M; kex = 280 nm; T = 296 K; pH = 7.4.
of the difenoconazole to HSA changed the microenvironment of tryptophan residue and the tertiary structure of HSA. 3.3. Binding parameters and mechanism A variety of molecular interactions can result in fluorescence quenching, including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching [17]. The different mechanisms of fluorescence quenching are usually classified as either dynamic quenching or static quenching. Dynamic quenching and static quenching are caused by diffusion and ground-state complex formation [17]. They have different dependence on temperature, higher temperature results in faster diffusion and larger amounts of collisional quenching. It will typically lead to the dissociation of weakly bound complexes and smaller amounts of static quenching. Therefore, the quenching constant increases for dynamic quenching while it decreases for static quenching with increase in temperature.
ð1Þ
where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. KQ the biomolecular quenching constant, s0 the life time of the fluorescence in absence of quencher (for most biomolecules, s0 is about 108 s), [Q] the concentration of quencher, and KSV is the Stern–Volmer quenching constant. The results summarized in Table 1 showed that the Stern–Volmer quenching constant KSV is inversely correlated with temperature and the values of KQ is greater than the limiting diffusion constant Kdif of the biomolecule (Kdif = 2.0 1010 L mol1 s1) [19]. This implied that the quenching was not initiated by dynamic collision but originated from the formation of a complex. The quantitative analysis of the binding of difenoconazole to HSA was carried out using the Scatchard’s procedure [20]
r=Df ¼ nK r K
ð2Þ
This method is based on the general equation, where r is the moles of molecule bound per mole of protein, Df is the molar concentration of free molecule, n is binding site multiplicity per class of binding sites, and K is the equilibrium binding constant. Fig. 8 shows the Scatchard plots for the system at different temperatures. The binding constants were summarized in Table 2. The binding constants K of Scatchard are approximate the former ones [21]. The linearity of Scatchard plots indicates that difenoconazole binds to one class of sites on HSA [22,23] and the binding constants decrease with the increasing temperature. It shows that the binding between difenoconazole and HSA is strong and the temperature has an effect on it. 3.4. Thermodynamic analysis There are four types of non-covalent interaction existing in ligand binding to protein. These are hydrogen bonds, van der Waals forces, hydrophobic and electrostatic interactions. The thermodynamic parameters, enthalpy and entropy of reaction, are important for confirming binding mode. For this purpose, the temperaturedependence of the binding constant was studied. The temperatures chosen were 296, 303, 310 and 317 K at which HSA does not undergo any structural degradation. By plotting the binding constants according to Van’t Hoff equation, the thermodynamic parameters were determined from a linear Van’t Hoff plot (spectrum not shown) and listed in Table 2. It is clear from the values of 4S and 4H that the binding of difenoconazole to HSA is an exothermic process accompanied by a positive values of 4S and a negative values of 4G. The binding process was always spontaneous as evidenced by the negative sign of 4G values. From the point of view of water structure [24], 4H > 0 and 4S > 0 implies a hydrophobic interaction; 4H < 0 and 4S < 0 reflects the van der Waals force or hydrogen bond formation; and 4H 0 and 4S > 0 suggests an electrostatic force. Therefore, the binding of difenoconazole to HSA might involve strongly hydrophobic interaction as verified by positive value of 4S while the electrostatic interaction
Table 1 The dynamic quenching constants between difenoconazole and HSA.
Fig. 7. Stern–Volmer plots of fluorescence quenching of difenoconazole–HSA system. [HSA] = 7.5 107 M; [difenoconazole] = 3.32 106 M–2.28 105 M; kex = 280 nm, kem = 350 nm; pH = 7.4.
T (K)
Stern–Volmer equation
KQ (L/mol/s)
R
296 K 303 K 310 K 317 K
Y = 1.02216 + 0.01257[Q] Y = 1.01799 + 0.01213[Q] Y = 1.01838 + 0.01124[Q] Y = 1.01774 + 0.01081[Q]
1.257 1012 1.213 1012 1.124 1012 1.081 1012
0.9989 0.9992 0.9981 0.9970
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and the absorption spectrum of difenoconazole can be calculated by the equation:
J¼
X
FðkÞeðkÞk4Dk=
X
FðkÞDk
ð3Þ
where F(k) is the fluorescence intensity of the fluorescent donor in wavelength k and is dimensionless, e(k) is the molar absorption coefficient of the acceptor in wavelength k. The critical distance R0 when the transfer efficiency is 50% can be obtained by plugging J into the following formula: 2
R60 ¼ 8:8 1025 k N4 U J
ð4Þ
2
where k is the spatial orientation factor of the dipole, N is the refractive index of the medium, U is the fluorescence quantum yield of the donor. The efficiency of energy transfer E is given by the following equation:
E ¼ 1 F=F 0 ¼ R60 =ðR60 þ r 6 Þ Fig. 8. The Scatchard curves of quenching of HSA with difenoconazole. [HSA] = 7.5 107 M; [difenoconazole] = 3.32 106 M–2.28 105 M; kex = 280 nm, kem = 350 nm; pH = 7.4.
Table 2 The binding constants and thermodynamic parameters between difenoconazole and HSA. T (K) 296 K 303 K 310 K 317 K
K (L/mol) 4
3.432 10 3.021 104 2.839 104 2.651 104
4G (kJ/mol)
4H (kJ/mol)
4S (J/mol K)
25.661 26.047 26.433 26.818
9.352
55.1
could not be excluded. That is, difenoconazole bound to HSA was mainly based on the hydrophobic and electrostatic interactions. 3.5. Binding distance According to Förster’s non-radiative energy transfer theory [25], the energy transfer will happen under the following conditions: (a) the donor can produce fluorescence light, (b) the extent of overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor and (c) the distance between the donor and the acceptor is lower than 7 nm. Because of the presence of tryptophan residues, HSA has strong fluorescence, and have more overlap with the UV absorption spectrum of difenoconazole (Fig. 9). The overlap integral of the fluorescence emission spectrum of HSA
Fig. 9. The overlap of fluorescence emission spectra of HSA (A) and UV–vis absorption spectra of difenoconazole (B). [HSA]: [difenoconazole] = 1:1.
ð5Þ
where r is the distance between acceptor and donor. Under these experimental conditions, the values of U, k2 and N were 0.118, 2/ 3 and 1.336, [26] respectively. Then the value of binding distance could be calculated to be 3.16 nm, which was lower than 7 nm, indicating the existence of the energy transfer between HSA to difenoconazole. 3.6. Molecule modeling of the complex of difenoconazole and HSA Human serum albumin is monomeric but contains three structurally similar a-helical domains (I–III); each domain has two subdomains (A and B), which are six (A) and four (B) a-helices, respectively. HSA has a limited number of binding sites for endogenous and exogenous ligands that are typically bound reversibly and have binding constants in the range 104–108 M1 [27]. The principal regions of ligand binding sites of albumin are located in hydrophobic cavities in subdomains IIA and IIIA, which exhibit similar chemistry. To establish which binding site of HSA that difenoconazole is located, the complementary applications of molecule modeling have been employed by computer methods to improve the understanding of the interaction of difenoconazole and HSA. The crystal structure of HSA in complex with R-warfarin was taken from the Brookhaven Protein Data Bank (entry codes 1h9z) [10]. Fig. 10 shows the best energy ranked results, difenoconazole molecule is located within the binding pocket of subdomain IIA. It can be seen that the hydrophobic cavity is large enough to accommodate the difenoconazole molecule. It is important to note that there
Fig. 10. The interaction model between difenoconazole and HSA. The secondary structure of the protein is shown and the important neighboring amino acids are labeled. Ligand structures are shown in a ‘‘ball and stick’’ format. The hydrogen bonds are indicated by red dash. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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are three hydrogen bonds between difenoconazole and Arg218 residues of HSA. The calculated binding Gibbs free energy (4G) is 21.20 kJ/mol, which is close to the experimental data (25.661 kJ/mol) in some degree. In addition, the Trp214 residue of HSA is in close proximity to difenoconazole, this finding provides a good structural basis to explain the efficient fluorescence quenching of HSA emission in the presence of difenoconazole [28]. 4. Conclusions The interaction of difenoconazole with HSA has been investigated in vitro under simulated physiological conditions (pH 7.4, ionic strength 0.1) using molecular modeling and different optical techniques. UV–vis absorption, FT-IR and CD spectrum studies revealed the changes in the secondary structure of HSA upon interaction with difenoconazole. A fluorescence method for the rapid and simple determination of the interaction between difenoconazole and HSA was provided. The binding constant is calculated from the fluorescence data. The molecular modeling study and thermodynamic analysis also suggested that difenoconazole could bind HSA through the hydrophobic force, electrostatic interaction and hydrogen bond between difenoconazole and HSA residue. This study presented here can be helpful for evaluation of toxicity of triazole antifungal agent and provide a theoretical basis for further research on other pesticide. Acknowledgment The presented study was supported by the National Natural Science Foundation of China (No. 51209068). References [1] M. Reuveni, D. Sheglov, Effects of azoxystrobin, difenoconazole, polyoxin B (polar) and trifloxystrobin on germination and growth of Alternaria alternata and decay in red delicious apple fruit, Crop Prot. 21 (2002) 951–955. [2] A.A. Ouameur, R. Marty, H.A. Tajmir-Riahi, Human serum albumin complexes with chlorophyll and chlorophyllin, Biopolymers 77 (2005) 129–136. [3] C. Ragi, M.M.R. Sedaghat-Herati, A.A. Ouameur, H.A. Tajmir-Riahi, The effects of poly(ethyleneglycol) on the solution structure of human serum albumin, Biopolymers 78 (2005) 231–236. [4] Y. Li, X.J. Yao, J. Jin, X.G. Chen, Z.D. Hu, Interaction of rhein with human serum albumin investigation by optical spectroscopic technique and modeling studies, Biochim. Biophys. Acta 1774 (2007) 51–58. [5] J. Chamani, A. Asoodeh, M. Homayoni-Tabrizi, Z.A. Tehranizadeh, A. Baratian, M.R. Saberi, M. Gharanfoli, Spectroscopic and nano-molecular modeling investigation on the binary and ternary bindings of colchicine and lomefloxacin to Human serum albumin with the viewpoint of multi-drug therapy, J. Lumin. 130 (2010) 2476–2486. [6] F.L. Cui, Q.Z. Zhang, X.J. Yao, H.X. Luo, Y. Yang, L.X. Qin, G.R. Qu, Y. Lu, The investigation of the interaction between 5-iodouracil and human serum albumin by spectroscopic and modeling methods and determination of protein by synchronous fluorescence technique, Pestic. Biochem. Physiol. 90 (2008) 126–134.
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Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Systematic investigation of the toxic mechanism of difenoconazole on protein by spectroscopic and molecular modeling Ying Li, Xueru Ma, Guanghua Lu ⇑ Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes of Ministry of Education, College of Environment, HoHai University, NanJing, JiangSu Province 210098, China
a r t i c l e
i n f o
Article history: Received 29 May 2012 Accepted 31 December 2012 Available online 20 January 2013 Keywords: Difenoconazole Protein Molecular modeling Multi-spectroscopy
a b s t r a c t In order to better understand the toxic effects of difenoconazole, we performed multi-spectroscopic measurements to quantify the interaction of difenoconazole with human serum albumin. The toxicity mechanism was predicted through molecular modeling, and the binding parameters were confirmed using a series of spectroscopic methods, including UV–vis absorption spectroscopy, Fourier transform infrared spectroscopy, circular dichroism spectroscopy and fluorescence spectroscopy. Alteration of secondary structure of protein was evaluated, then the specific binding site in protein was identified and binding constants were determined. The molecular modeling study and thermodynamic analysis suggested that difenoconazole could bind HSA through the hydrophobic force, electrostatic interaction and hydrogen bond. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Difenoconazole (structure shown in Fig. 1) was developed by Switzerland Syngenta Corporation, IUPAC name: cis,trans-3chloro-4-[4-methyl-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan2-yl] phenyl-4-chlorophenylether)phenyl-4-chlorophenyl ether, belonging to the triazole group of pesticides. As a kind of systemic sterol demethylation inhibitor (DMI), difenoconazole has a good ability to interfere with the mycelial growth and inhibit the spore germination of pathogens that ultimately results in inhibiting fungal growth [1]. Difenoconazole is extensively used in a wide range of crops in many countries for its good control of various fungal diseases. However most of pesticides are not completely degraded after application, their metabolites and some unchanged forms of these compounds are excreted and subsequently enter the ecosystem. Although the triazole fungicides have shorter half-lives and lower bioaccumulation than the organochlorine pesticides, possible detrimental effects on the ecosystem and human health were also existed. For this reason, knowledge of the interaction mechanisms between difenoconazole and plasma protein is of crucial importance for us to understand its possible danger to human body. On the basis of the motivations described above, we settled on an initial focus on difenoconazole binding to basic human plasma protein, and understanding the toxic response. Human serum albumin (HSA) is the most abundant protein in the blood, which is ⇑ Corresponding author. E-mail address: [email protected] (G. Lu). 0048-3575/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2012.12.010
globular protein composed of a single polypeptide chain of 585 amino acid residues. It is characterized by high a-helical content and a large number of disulfide bonds, and serves in transportation and disposition of many endogenous substances and exogenous compounds, including drugs and xenobiotics [2–7]. A wide range of photophysical techniques, such as fluorescence quenching, circular dichriosm (CD) and Fourier transform infrared (FT-IR) are used in our study to characterize various aspects of the protein–difenoconazole interaction and these techniques are briefly described in Section 2. These measurement methods allow for determination of a number of basic properties: the binding constant of proteins with difenoconazole, changes in protein conformation. Specifically, fluorescence quenching measurements give information about the protein–difenoconazole binding kinetics and equilibrium and protein conformational change, CD and FTIR informs about changes in protein structure upon binding, and molecular modeling investigate the mechanism of the reaction. The use of these combined methods should provide a general perspective of how difenoconazole affects the nature of protein binding and the extent to which these effects are protein specific. These supporting information are very important for food safety and human health when using triazole antifungal agent. 2. Materials and methods 2.1. Materials Difenoconazole was of chromatographic grade, and purchased from Dr. Ehrenstorfer Company (Germany). Human Serum
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O
Cl
Cl
O
experiments were performed at four different temperatures (296, 303, 310 and 317 K). O
N N N
CH 3 Fig. 1. Chemical structure of difenoconazole.
Albumin (HSA, 70024-90-7, 98% purity) was purchased from Sigma Chemical Company. It was used without further purification and its molecular weight was assumed to be 66,478 to calculate molar concentrations. NaCl (1.0 mol/L) solution was used to maintain the ionic strength at 0.1. Buffer consists of Tris (0.2 mol/L) and HCl (0.1 mol/L), and the pH was adjusted to 7.40. A HSA solution of 1.5 105 mol/L was prepared using the Tris–HCl buffer solution and kept in dark at 4 °C. The stock solution of difenoconazole (1.0 103 mol/L) was prepared in absolute ethanol. All the other reagents were of analytical grade and doubly distilled water was used throughout the experiment.
2.2. Apparatus and methods 2.2.1. Multi-spectroscopic measurements Infrared spectroscopy were recorded at 296 K using a Nicolet Nexus 670 FT-IR Spectrometer (USA) equipped with a Geranium attenuated total reflection (ATR) accessory, a deuterated triglycine sulphate (DTGS) detector and a KBr beam splitter. All infrared spectra were taken via the attenuated total reflection method with a resolution of 4 cm1 and 128 scans. FT-IR spectrum of free HSA was acquired by subtracting the absorption of the Tris buffer solution from the spectrum of the protein solution, and a difference spectrum of HSA was obtained by subtracting the spectrum of difenoconazole from that of difenoconazole–HSA with the same concentration. The subtraction criterion was that original spectrum of the protein solution between 2200 and 1800 cm1 was featureless [8]. Secondary derivative were applied to this range respectively to estimate the number, position and width of component bands. Based on these parameters curve-fitting process was carried out by Galactic Peak solve to get the best Gaussian-shaped curves that fit the original protein spectrum. After the identification of the individual bands, the representative structure of HSA was calculated using the area of their respective component bands. Circular dichroism measurements were made on a Chriascan CD Spectrometric (Applied photophysics, UK). CD spectra were recorded in the range of 200–250 nm. The a-helical content of HSA was calculated from the molar ellipticity [h] at 208 nm using the following equation [9]:
2.2.2. Molecular modeling study The crystal structure of HSA in complex with R-warfarin was taken from the Brookhaven Protein Data Bank (entry codes 1h9z) [10]. The potential of the 3D structure of HSA was assigned based on the Amber 4.0 force field with Kollman-all-atom charges. The initial structure of difenoconazole was generated by molecular modeling software Sybyl 6.9 [11]. The geometry of the molecule was subsequently optimized to minimal energy using the Tripos force field with Gasteiger–Marsili charges, and the FlexX program was used to build the interaction modes between difenoconazole and HSA. All calculations were performed on a SGI FUEL workstation. 3. Results and discussion 3.1. Binding studies between difenoconazole and HSA using UV–vis absorption, FT-IR and CD spectrum In order to gain a better understanding in physicochemical properties of difenoconazole governing its spectral behavior and to draw relevant conclusions on the difenoconazole–HSA binding mechanism UV–vis absorption, FT-IR and CD spectroscopic measurements were performed on HSA and the difenoconazole–HSA complex. If the change of protein structure included the transforming of protein secondary structure in the complex, it can be reflected in the spectra. Fig. 2 showed the UV–vis absorption spectra of HSA in the absence and presence of difenoconazole. It can be seen from Fig. 2, the absorbance of HSA increased upon the addition of difenoconazole. At the same time, the maximum peak position of difenoconazole–HSA was shifted slightly towards lower wavelength region. This indicated the change in polarity around the tryptophan residue and the change in peptide strand of HSA molecules and hence the change in hydrophobicity [12]. These two results clearly indicated that bound to HSA and the conformation of HSA was changed. Infrared spectroscopy has long been used as a powerful method for investigating the secondary structures of proteins and their dynamics. In the IR region, the frequencies of bands due to the
a helixð%Þ ¼ fð½h208 4000Þ=ð33000 4000Þg 100 UV–vis absorption spectra were recorded on a UV-3600 UV– VIS–NIR spectrophotometer (SHIMADZU, Japan) equipped with 1.0 cm quartz cells. The wavelength range was 200–500 nm. The Fluorescence spectra were measured with a LS-50B spectrofluorophotometer (PERKIN-ELMER, USA). The fluorescence excitation wavelength was 280 nm, and the emission was read at 300– 500 nm. In a typical fluorometric titration experiment: 3.0 mL solution containing an appropriate concentration of HSA was titrated in the Tris buffer solution by successive additions of difenoconazole working solution. Titrations were done manually using micropipettes, and the fluorescence intensities were recorded at excitation wavelength of 280 nm and emission wavelength of 350 nm. All
Fig. 2. UV–vis absorption spectra of difenoconazole–HSA system. (A) [difenoconazole] = 3.32 106 M; (B) [HSA] = 1.5 106 M; (C–E) difenoconazole–HSA; [HSA] = 1.5 106 M; [difenoconazole] = 3.32 106 M, 6.62 106 M, 1.32 105 M; T = 296 K; pH = 7.4.
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amide I–III vibrations are sensitive to the secondary structure of proteins. The amide I peak position occurs in the region 1600– 1700 cm1 and the amide II band 1548 cm1 [13,14]. In the amide I region, the observed stretching frequency of C@O hydrogen bonded to NH moieties is dependent upon the secondary structure adopted by the peptide chain. Changes in the structure of the protein are reflected by changes in the component band positions of the amide I region [15]. Fig. 3 shows the FT-IR spectra of free HSA and its difenoconazole complexes in Tris–HCl buffer solution at 296 K. As shown in Fig. 3, the peak position of amide I of HSA moved from 1650 to 1640 cm1 and the peak position of amide II of HSA shifted from 1550 to 1583 cm1 after addition of difenoconazole. The changes of this peak position demonstrated that difenoconazole interacted with HSA and caused a change in the secondary structure of HSA. According to the curve-fitted results (Fig. 4), the secondary structure compositions of HSA in the absence and in the presence of difenoconazole were estimated. As shown in Fig. 4, the content of b-sheet structure composition of HSA increased from 33.15% to 39.08%, the content of b-turn structure increased from 30.72% to 33.15%, while the content of a-helix structure decreased from 36.13% to 27.77%. CD spectra of HSA exhibit characteristic features of the a-helical structure of protein with negative bands in the ultraviolet region at 208 and 222 nm [16]. Here the CD spectrum was taken in the wavelength range from 200 to 250 nm. Fig. 5 shows typical CD spectrum of HSA in the presence and absence of difenoconazole. The binding of difenoconazole complex to HSA decrease the ellipticity values in the CD spectrum, indicating the considerable changes in the protein secondary structure with the reduction of the a-helical content in HSA and the increase of the disorder structure content in the protein. The calculating results exhibited a reduction of a-helix structures from 52.98% to 48.23%. Decrease in the a-helical content in the CD spectra is consistent with the IR results. 3.2. Analysis of fluorescence quenching of HSA by difenoconazole
Fig. 4. The curve fitting amide I region with secondary structure (1700–1600 cm1). (A) HSA; (B) difenoconazole–HSA; [HSA] = 6.0 105 M; [difenoconazole] = 3.0 104 M; pH = 7.4.
An intrinsic fluorescence study was performed to evaluate changes in tertiary structure caused by reaction of HSA with difenoconazole. The effect of difenoconazole on HSA fluorescence intensity is shown in Fig. 6. The addition of difenoconazole caused a gradual decrease in the fluorescence emission intensity of HSA
Fig. 5. CD Spectra of the difennoconazole–HSA System. (A) HSA; (B) difenoconazole–HSA; [HSA] = 6.0 107 M; [difenoconazole] = 3.0 105 M; T = 296 K; pH = 7.4.
Fig. 3. FT-IR spectra and difference spectra of HSA in aqueous solution. (A) FT-IR spectrum of free HSA; (B) FT-IR difference spectrum [(HSA solution + difenoconazole solution)(difenoconazole solution)] in buffer solution; [HSA] = 6.0 105 M; [difenoconazole] = 3.0 104 M; T = 296 K; pH = 7.4.
with a conspicuous change in the emission spectra. It can be seen that a higher excess of difenoconazole led to more effective quenching of the chromophore molecule fluorescence. The strong quenching of the fluorescence clearly indicated that the binding
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The possible quenching mechanism can be deduced from the Stern–Volmer plot (Fig. 7). The classical Stern–Volmer equation [18]:
F0 ¼ 1 þ K Q s0 ½Q ¼ 1 þ K SV ½Q F
Fig. 6. Fluorescence emission spectra of difenoconazole–HSA system. (A) HSA; (B– H) difenoconazole–HSA; [HSA] = 7.5 107 M; [difenoconazole] = 3.32 106 M, 6.62 106 M, 9.90 106 M, 1.32 105 M, 1.64 105 M, 1.96 105 M, 2.28 105 M; kex = 280 nm; T = 296 K; pH = 7.4.
of the difenoconazole to HSA changed the microenvironment of tryptophan residue and the tertiary structure of HSA. 3.3. Binding parameters and mechanism A variety of molecular interactions can result in fluorescence quenching, including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching [17]. The different mechanisms of fluorescence quenching are usually classified as either dynamic quenching or static quenching. Dynamic quenching and static quenching are caused by diffusion and ground-state complex formation [17]. They have different dependence on temperature, higher temperature results in faster diffusion and larger amounts of collisional quenching. It will typically lead to the dissociation of weakly bound complexes and smaller amounts of static quenching. Therefore, the quenching constant increases for dynamic quenching while it decreases for static quenching with increase in temperature.
ð1Þ
where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. KQ the biomolecular quenching constant, s0 the life time of the fluorescence in absence of quencher (for most biomolecules, s0 is about 108 s), [Q] the concentration of quencher, and KSV is the Stern–Volmer quenching constant. The results summarized in Table 1 showed that the Stern–Volmer quenching constant KSV is inversely correlated with temperature and the values of KQ is greater than the limiting diffusion constant Kdif of the biomolecule (Kdif = 2.0 1010 L mol1 s1) [19]. This implied that the quenching was not initiated by dynamic collision but originated from the formation of a complex. The quantitative analysis of the binding of difenoconazole to HSA was carried out using the Scatchard’s procedure [20]
r=Df ¼ nK r K
ð2Þ
This method is based on the general equation, where r is the moles of molecule bound per mole of protein, Df is the molar concentration of free molecule, n is binding site multiplicity per class of binding sites, and K is the equilibrium binding constant. Fig. 8 shows the Scatchard plots for the system at different temperatures. The binding constants were summarized in Table 2. The binding constants K of Scatchard are approximate the former ones [21]. The linearity of Scatchard plots indicates that difenoconazole binds to one class of sites on HSA [22,23] and the binding constants decrease with the increasing temperature. It shows that the binding between difenoconazole and HSA is strong and the temperature has an effect on it. 3.4. Thermodynamic analysis There are four types of non-covalent interaction existing in ligand binding to protein. These are hydrogen bonds, van der Waals forces, hydrophobic and electrostatic interactions. The thermodynamic parameters, enthalpy and entropy of reaction, are important for confirming binding mode. For this purpose, the temperaturedependence of the binding constant was studied. The temperatures chosen were 296, 303, 310 and 317 K at which HSA does not undergo any structural degradation. By plotting the binding constants according to Van’t Hoff equation, the thermodynamic parameters were determined from a linear Van’t Hoff plot (spectrum not shown) and listed in Table 2. It is clear from the values of 4S and 4H that the binding of difenoconazole to HSA is an exothermic process accompanied by a positive values of 4S and a negative values of 4G. The binding process was always spontaneous as evidenced by the negative sign of 4G values. From the point of view of water structure [24], 4H > 0 and 4S > 0 implies a hydrophobic interaction; 4H < 0 and 4S < 0 reflects the van der Waals force or hydrogen bond formation; and 4H 0 and 4S > 0 suggests an electrostatic force. Therefore, the binding of difenoconazole to HSA might involve strongly hydrophobic interaction as verified by positive value of 4S while the electrostatic interaction
Table 1 The dynamic quenching constants between difenoconazole and HSA.
Fig. 7. Stern–Volmer plots of fluorescence quenching of difenoconazole–HSA system. [HSA] = 7.5 107 M; [difenoconazole] = 3.32 106 M–2.28 105 M; kex = 280 nm, kem = 350 nm; pH = 7.4.
T (K)
Stern–Volmer equation
KQ (L/mol/s)
R
296 K 303 K 310 K 317 K
Y = 1.02216 + 0.01257[Q] Y = 1.01799 + 0.01213[Q] Y = 1.01838 + 0.01124[Q] Y = 1.01774 + 0.01081[Q]
1.257 1012 1.213 1012 1.124 1012 1.081 1012
0.9989 0.9992 0.9981 0.9970
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and the absorption spectrum of difenoconazole can be calculated by the equation:
J¼
X
FðkÞeðkÞk4Dk=
X
FðkÞDk
ð3Þ
where F(k) is the fluorescence intensity of the fluorescent donor in wavelength k and is dimensionless, e(k) is the molar absorption coefficient of the acceptor in wavelength k. The critical distance R0 when the transfer efficiency is 50% can be obtained by plugging J into the following formula: 2
R60 ¼ 8:8 1025 k N4 U J
ð4Þ
2
where k is the spatial orientation factor of the dipole, N is the refractive index of the medium, U is the fluorescence quantum yield of the donor. The efficiency of energy transfer E is given by the following equation:
E ¼ 1 F=F 0 ¼ R60 =ðR60 þ r 6 Þ Fig. 8. The Scatchard curves of quenching of HSA with difenoconazole. [HSA] = 7.5 107 M; [difenoconazole] = 3.32 106 M–2.28 105 M; kex = 280 nm, kem = 350 nm; pH = 7.4.
Table 2 The binding constants and thermodynamic parameters between difenoconazole and HSA. T (K) 296 K 303 K 310 K 317 K
K (L/mol) 4
3.432 10 3.021 104 2.839 104 2.651 104
4G (kJ/mol)
4H (kJ/mol)
4S (J/mol K)
25.661 26.047 26.433 26.818
9.352
55.1
could not be excluded. That is, difenoconazole bound to HSA was mainly based on the hydrophobic and electrostatic interactions. 3.5. Binding distance According to Förster’s non-radiative energy transfer theory [25], the energy transfer will happen under the following conditions: (a) the donor can produce fluorescence light, (b) the extent of overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor and (c) the distance between the donor and the acceptor is lower than 7 nm. Because of the presence of tryptophan residues, HSA has strong fluorescence, and have more overlap with the UV absorption spectrum of difenoconazole (Fig. 9). The overlap integral of the fluorescence emission spectrum of HSA
Fig. 9. The overlap of fluorescence emission spectra of HSA (A) and UV–vis absorption spectra of difenoconazole (B). [HSA]: [difenoconazole] = 1:1.
ð5Þ
where r is the distance between acceptor and donor. Under these experimental conditions, the values of U, k2 and N were 0.118, 2/ 3 and 1.336, [26] respectively. Then the value of binding distance could be calculated to be 3.16 nm, which was lower than 7 nm, indicating the existence of the energy transfer between HSA to difenoconazole. 3.6. Molecule modeling of the complex of difenoconazole and HSA Human serum albumin is monomeric but contains three structurally similar a-helical domains (I–III); each domain has two subdomains (A and B), which are six (A) and four (B) a-helices, respectively. HSA has a limited number of binding sites for endogenous and exogenous ligands that are typically bound reversibly and have binding constants in the range 104–108 M1 [27]. The principal regions of ligand binding sites of albumin are located in hydrophobic cavities in subdomains IIA and IIIA, which exhibit similar chemistry. To establish which binding site of HSA that difenoconazole is located, the complementary applications of molecule modeling have been employed by computer methods to improve the understanding of the interaction of difenoconazole and HSA. The crystal structure of HSA in complex with R-warfarin was taken from the Brookhaven Protein Data Bank (entry codes 1h9z) [10]. Fig. 10 shows the best energy ranked results, difenoconazole molecule is located within the binding pocket of subdomain IIA. It can be seen that the hydrophobic cavity is large enough to accommodate the difenoconazole molecule. It is important to note that there
Fig. 10. The interaction model between difenoconazole and HSA. The secondary structure of the protein is shown and the important neighboring amino acids are labeled. Ligand structures are shown in a ‘‘ball and stick’’ format. The hydrogen bonds are indicated by red dash. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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are three hydrogen bonds between difenoconazole and Arg218 residues of HSA. The calculated binding Gibbs free energy (4G) is 21.20 kJ/mol, which is close to the experimental data (25.661 kJ/mol) in some degree. In addition, the Trp214 residue of HSA is in close proximity to difenoconazole, this finding provides a good structural basis to explain the efficient fluorescence quenching of HSA emission in the presence of difenoconazole [28]. 4. Conclusions The interaction of difenoconazole with HSA has been investigated in vitro under simulated physiological conditions (pH 7.4, ionic strength 0.1) using molecular modeling and different optical techniques. UV–vis absorption, FT-IR and CD spectrum studies revealed the changes in the secondary structure of HSA upon interaction with difenoconazole. A fluorescence method for the rapid and simple determination of the interaction between difenoconazole and HSA was provided. The binding constant is calculated from the fluorescence data. The molecular modeling study and thermodynamic analysis also suggested that difenoconazole could bind HSA through the hydrophobic force, electrostatic interaction and hydrogen bond between difenoconazole and HSA residue. This study presented here can be helpful for evaluation of toxicity of triazole antifungal agent and provide a theoretical basis for further research on other pesticide. Acknowledgment The presented study was supported by the National Natural Science Foundation of China (No. 51209068). References [1] M. Reuveni, D. Sheglov, Effects of azoxystrobin, difenoconazole, polyoxin B (polar) and trifloxystrobin on germination and growth of Alternaria alternata and decay in red delicious apple fruit, Crop Prot. 21 (2002) 951–955. [2] A.A. Ouameur, R. Marty, H.A. Tajmir-Riahi, Human serum albumin complexes with chlorophyll and chlorophyllin, Biopolymers 77 (2005) 129–136. [3] C. Ragi, M.M.R. Sedaghat-Herati, A.A. Ouameur, H.A. Tajmir-Riahi, The effects of poly(ethyleneglycol) on the solution structure of human serum albumin, Biopolymers 78 (2005) 231–236. [4] Y. Li, X.J. Yao, J. Jin, X.G. Chen, Z.D. Hu, Interaction of rhein with human serum albumin investigation by optical spectroscopic technique and modeling studies, Biochim. Biophys. Acta 1774 (2007) 51–58. [5] J. Chamani, A. Asoodeh, M. Homayoni-Tabrizi, Z.A. Tehranizadeh, A. Baratian, M.R. Saberi, M. Gharanfoli, Spectroscopic and nano-molecular modeling investigation on the binary and ternary bindings of colchicine and lomefloxacin to Human serum albumin with the viewpoint of multi-drug therapy, J. Lumin. 130 (2010) 2476–2486. [6] F.L. Cui, Q.Z. Zhang, X.J. Yao, H.X. Luo, Y. Yang, L.X. Qin, G.R. Qu, Y. Lu, The investigation of the interaction between 5-iodouracil and human serum albumin by spectroscopic and modeling methods and determination of protein by synchronous fluorescence technique, Pestic. Biochem. Physiol. 90 (2008) 126–134.
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